SUMMARY We report that t(1;19)-ALL cells universally exhibit expression of and dependence on the cell surface receptor, ROR1. We further identify t(1;19)-ALL cell sensitivity to the kinase inhibitor dasatinib due to its inhibition of the pre-B-cell receptor (pre-BCR) signaling complex. These phenotypes are a consequence of developmental arrest at an intermediate/late stage of B-lineage maturation. Additionally, inhibition of pre-BCR signaling induces further ROR1 upregulation, and we identify distinct ROR1 and pre-BCR downstream signaling pathways that are modulated in a counter-balancing manner—both leading to AKT phosphorylation. Consistent with this, AKT phosphorylation is transiently eliminated after dasatinib treatment, but is partially restored following dasatinib potentiation of ROR1 expression. Consequently, ROR1 silencing accentuates dasatinib killing of t(1;19)-ALL cells.

Methylation of histone H3 at lysine 36 (H3K36me), a widely-distributed chromatin mark, largely results from association of the lysine methyltransferase (KMT) SET-2 with RNA polymerase II (RNAPII), but most eukaryotes also have additional H3K36me KMTs that act independently of RNAPII. These include the orthologs of ASH1, which are conserved in animals, plants, and fungi but whose function and control are poorly understood. We found that Neurospora crassa has just two H3K36 KMTs, ASH1 and SET-2, and were able to explore the function and distribution of each enzyme independently. While H3K36me deposited by SET-2 marks active genes, inactive genes are modified by ASH1 and its activity is critical for their repression. ASH1-marked chromatin can be further modified by methylation of H3K27, and ASH1 catalytic activity modulates the accumulation of H3K27me2/3 both positively and negatively. These findings provide new insight into ASH1 function, H3K27me2/3 establishment, and repression in facultative heterochromatin.

DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1–associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation.

A decrease in reactive oxygen species (ROS) production has been associated with extended life span in animal models of longevity. Mice deficient in the p66Shc gene are long-lived, and their cells are both resistant to oxidative stress and produce less ROS. Our microarray analysis of p66Shc(؊/؊) mouse tissues showed alterations in transcripts involved in heme and superoxide production and insulin signaling. Thus, we carried out analysis of ROS production by NADPH oxidase (PHOX) in macrophages of control and p66Shc knock-out mice. p66Shc(؊/؊) mice had a 40% reduction in PHOX-dependent superoxide production. To confirm whether the defect in superoxide production was a direct consequence of p66Shc deficiency, p66Shc was knocked down with siRNA in the macrophage cell line RAW264, and a 30% defect in superoxide generation was observed. The pathway of PHOX-dependent superoxide generation was investigated. PHOX protein levels were not decreased in mutant macrophages; however, the rate and extent of phosphorylation of p47phox was decreased in mutants, as was membrane translocation of the complex. Consistently, phosphorylation of protein kinase C␦, Akt, and ERK (the kinases responsible for phosphorylation of p47phox) was decreased. Thus, p66Shc deficiency causes a defect in activation of the PHOX complex that results in decreased superoxide production. p66Shc-deficient mice have recently been observed to be resistant to atherosclerosis and to oxidant injury in kidney and brain. Because phagocyte-derived superoxide is often a component of oxidant injury and inflammation, we suggest that the decreased superoxide production by PHOX in p66Shc-deficient mice could contribute significantly to their relative protection from oxidant injury and consequent longevity.

The salts [Pt{C(NHMe)(2)}(4)][Au(CN)(2)](2), [Pt{C(NHMe)(2)}(4)][Ag(2)(CN)(3)][Ag(CN)(2)], [Pt(en)(2)][Au(CN)(2)](2), [Pt(en)(2)][Ag(CN)(2)](2), and [Pt(bipy)(2)][Au(CN)(2)](2) have been prepared by mixing solutions of salts containing the appropriate cation with solutions of K[Au(CN)(2)] or K[Ag(CN)(2)]. Because the platinum atom in the cation is sterically protected, the structures of [Pt{C(NHMe)(2)}(4)][Au(CN)(2)](2) and [Pt{C(NHMe)(2)}(4)][Ag(2)(CN)(3)][Ag(CN)(2)] reveal no close metal-metal interactions. Colorless crystals of [Pt(en)(2)][Au(CN)(2)](2) and [Pt(en)(2)][Ag(CN)(2)](2) are isostructural and involve extended chains of alternating cations and anions that run parallel to the crystallographic a axis, along with isolated anions. In the chains, the metal-metal separations are relatively short: Pt...Au, 3.1799(3) Angstroms; Pt...Ag, 3.1949(2) Angstroms. In [Pt(bipy)(2)][Au(CN)(2)](2), each cation has axial interactions with the anions through close Pt...Au contacts [3.1735(6) Angstroms]. In addition, the anions are weakly linked through Au...Au contacts of 3.5978(9) Angstroms. Unlike the previously reported Pt/Au complex [Pt(NH(3))(4)][Au(CN)(2)](2).1.5H(2)O, which is luminescent, none of the salts reported here luminesce.